www.nature.com/scientificreports OPEN Stingless bee honey, a novel source of trehalulose: a biologically active disaccharide with health benefts Mary T. Fletcher 1*, Natasha L. Hungerford 1, Dennis Webber2, Matheus Carpinelli de Jesus 3, Jiali Zhang1, Isobella S. J. Stone 2,3, Joanne T. Blanchfeld 3 & Norhasnida Zawawi 1,4* Stingless bee (Meliponini) honey has long been considered a high-value functional food, but the perceived therapeutic value has lacked attribution to specifc bioactive components. Examination of honey from fve diferent stingless bee species across Neotropical and Indo-Australian regions has enabled for the frst time the identifcation of the unusual disaccharide trehalulose as a major component representing between 13 and 44 g per 100 g of each of these honeys. Trehalulose is an isomer of sucrose with an unusual α-(1 → 1) glucose-fructose glycosidic linkage and known acariogenic and low glycemic index properties. NMR and UPLC-MS/MS analysis unambiguously confrmed the identity of trehalulose isolated from stingless bee honeys sourced across three continents, from Tetragonula carbonaria and Tetragonula hockingsi species in Australia, from Geniotrigona thoracica and Heterotrigona itama in Malaysia and from Tetragonisca angustula in Brazil. The previously unrecognised abundance of trehalulose in stingless bee honeys is concrete evidence that supports some of the reported health attributes of this product. This is the frst identifcation of trehalulose as a major component within a food commodity. This study allows the exploration of the expanded use of stingless bee honey in foods and identifes a bioactive marker for authentication of this honey in associated food standards. Stingless bees (Meliponini) occur in most tropical and sub-tropical regions, with over 500 species of these eusocial bees distributed across Neotropical, Afrotropical and Indo-Australian regions 1. Like the more well- recognised Apis mellifera honeybees, stingless bees live in permanent colonies made up of a single queen and workers, who collect pollen and nectar to feed larvae within the colony and likewise store honey in the hive for this purpose. Honey produced by stingless bees is known by various names such as Meliponine honey, pot- honey, sugarbag honey (in Australia), and Kelulut honey (in Malaysia). Under these and other names, stingless bee honey has a long history of traditional indigenous use with a range of purported therapeutic properties2, including antidiabetic and antioxidant activity3,4. Various studies have been conducted of physicochemical and nutritional composition of stingless bee honey5, but to date few bioactive components have been identifed2. While these studies all acknowledged that the composition of stingless bee honey is diferent to that of European bee honey, no rigorous identifcation of the major components and potential therapeutically active compounds has been reported. In addition to the importance of identifying the potential therapeutic components of stingless bee honey, the rapidly increasing consumer demand for stingless bee honey derived products has highlighted the need to produce food standards to enable establishment of authenticity and provenance of such products6. We report here for the frst time that the disaccharide trehalulose (1) (Fig. 1) is a major component of sting- less bee honeys from Malaysia, Australia and Brazil. Trehalulose is a naturally occurring isomer of sucrose, but has a much slower rate of release of monosaccharides into the bloodstream than sucrose7,8. Tis disaccharide is therefore highly benefcial in having both a low insulinemic index and low glycemic index9. Trehalulose is 1Queensland Alliance for Agriculture and Food Innovation (QAAFI), The University of Queensland, Brisbane, QLD 4072, Australia. 2Biosecurity Queensland, Department of Agriculture and Fisheries, Brisbane, QLD 4108, Australia. 3School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, QLD 4072, Australia. 4Department of Food Science, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia. *email: [email protected]; [email protected] SCIENTIFIC REPORTS | (2020) 10:12128 | https://doi.org/10.1038/s41598-020-68940-0 1 Vol.:(0123456789) www.nature.com/scientificreports/ Figure 1. Chemical structures of trehalulose (1) (major, fructopyranose (1a) and minor, fructofuranose (1b) tautomers), maltose (2) and isomaltulose (palatinose) (3). also known to be acariogenic 10,11, and a highly active antioxidant 12, and these properties may in no small way contribute to the reported benefcial health properties of stingless bee honey. Results Our UPLC-MS/MS analysis of honey from each of the fve stingless bee species studied showed the presence of fructose, glucose and a single major disaccharide, the same disaccharide in each of the honeys regardless of species. From HRMS data, this major component with molecular ion ([M-H]−) m/z 341.1082 (calculated for − [C12H22O11-H] : 341.1089) was clearly a disaccharide, but did not match any of our initially available disaccha- ride standards. Previous analysis of stingless bee honeys have suggested that the disaccharide present was the glucose-glucose disaccharide maltose (2)13–18. However, the improved resolution and mass spectral data provided by our UPLC-MS/MS method demonstrated that the disaccharide present in all fve stingless bee honeys, was in fact not maltose. Tis enigmatic disaccharide eluted with a slightly shorter UPLC retention time compared to a maltose standard (Fig. 2), and the mass fragmentation (MS/MS) difered from that of maltose (Fig. 3). Indeed examination of MS/MS fragmentation confrmed that it was instead a glucose-fructose disaccharide. We have subsequently isolated the disaccharide present in samples of honey from each of the fve stingless bee species by preparative HPLC and confrmed the identity of the disaccharide as trehalulose (1) (Fig. 1), a biologically active disaccharide that has not previously been reported in stingless bee honey. NMR characterisation of trehalulose. 1D and 2D NMR analysis of the isolated disaccharide suggested a 1,1-linkage between glucose and fructose monosaccharides with evidence of major/minor tautomers for the fructose ring (1a) and (1b). Tentative structural assignments were compared with literature enabling the unam- biguous assignment of the unknown honey disaccharide as trehalulose (Fig. 1). Trehalulose is one of only a few oligosaccharides in which the fructose ring exists predominantly in the pyranose form in solution with the 1-O-α-d-glucopyranosyl-β-d-fructopyranose tautomer (1a) being present at a higher level than 1-O-α-d- glucosylpyranosyl-β-d-fructofuranose tautomer (1b)19. Trehalulose was previously isolated from excrement of sweet potato whitefy Bemisia tabaci by Bates et al.20 Tese authors reported both 13C and 1H NMR assignments in D2O of three tautomers present in 20:4:1 ratio. Teir NMR data were similar to incomplete details reported previously by Cookson 19, who had isolated trehalulose from a mixture of glucose, fructose and isomaltulose produced by immobolised microbial cells, with the isolated disaccharide reported to be a 2:1 mixture of fructo- pyranose and fructofuranose forms by 13C NMR and other methods. Te 1D and 2D NMR obtained in this study confrmed that our isolated trehalulose was a mixture of both fructopyranose/fructofuranose tautomers (1a) and (1b), which were present in a 3.6:1 ratio by integration of the anomeric proton signals for the glucose rings (Table 1). Te anomeric glucosyl proton signal H1′ (4.98 ppm) and fructosyl H6a (4.08 ppm) of the major fructopyranose conformer (1a) were irradiated in separate 1D TOCSY experiments to identify the proton signals in each monosaccharide spin system. Tis allowed full elucidation of the pyranose tautomer of fructose (β-d-Frup) predominant in trehalulose (1), with assigned 13C and 1H NMR data of (1a) shown to be in close agreement to that previously reported by Bates et al.20 for this compound (Table 1) and more recently depicted by Garcia-Gonzalez et al.21. 1H NMR resonances for the minor fructofuranose tautomer (1b) were partly obscured by the more domi- nant fructopyranose tautomer (1a) in the isolated trehalulose. Only the anomeric glucosyl proton doublet (H1′ 5.01 ppm, 3.7 Hz) could be clearly distinguished. Based on 2D HSQC, TOCSY and COSY experiments, all other protons and carbon shifs of the minor fructofuranose conformer (1b) were assigned as shown in Table 1. Tis full assignment extends the previous partial assignment reported by Bates et al.20. A third more minor tautomer with anomeric glucosyl proton doublet (H1′ 5.00 ppm, 3.7 Hz) could be seen in our trehalulose NMR spectra, but due to the low concentration and overlap with the other anomeric signals it is not possible to report full data for this compound. Tis is presumably the component speculated to be the α-furanose tautomer by Bates et al.20. Close examination of NMR spectra demonstrated that the disaccharide isolated from each of the fve stingless bees comprised > 90% trehalulose (1) with minor disaccharides tentatively identifed as sucrose, maltulose and turanose by comparison of NMR chemical shifs with literature 22. Isomaltulose (3)22 was not detected in these NMR spectra of trehalulose isolated from stingless bee honey. SCIENTIFIC REPORTS | (2020) 10:12128 | https://doi.org/10.1038/s41598-020-68940-0 2 Vol:.(1234567890) www.nature.com/scientificreports/ Figure 2. UPLC-MS/MS selected-ion chromatograms